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First published online July 22, 2005; 10.1104/pp.105.063537 Plant Physiology 138:1842-1852 (2005) © 2005 American Society of Plant Biologists An RNA-Dependent RNA Polymerase Prevents Meristem Invasion by Potato Virus X and Is Required for the Activity But Not the Production of a Systemic Silencing Signal1,[w]Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom
One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.
Endogenous RNA-dependent RNA polymerase (RDR) activity was demonstrated in plants more than 30 years ago (Astier-Manifacier and Cornuet, 1974  ), but its function remained unclear for many years. More recently, RDRs were proposed as part of an RNA silencing system (Lindbo et al., 1993) related to RNA interference in animals and quelling in fungi. Consistent with this idea, the SILENCING DEFECTIVE 1 (SDE1)/SUPPRESSOR OF GENE SILENCING 2(SGS2) locus was identified as a requirement for certain examples of transgene RNA silencing in Arabidopsis (Arabidopsis thaliana; Dalmay et al., 2000; Mourrain et al., 2000). Following a more recent nomenclature, we will henceforth refer to SDE1/SGS2 as RDR6 (Xie et al., 2004).
A likely biochemical role of RDRs in RNA silencing is to produce double-stranded (ds) RNA that is cleaved by RNase III-like enzymes called Dicer (DCR) in animals and Dicer-like (DCL) in plants (Bernstein et al., 2001
There are several RNA silencing pathways (Baulcombe, 2004
An additional manifestation of RNA silencing that is associated with virus defense in plants is a signal that spreads through the plasmodesmata and phloem (Palauqui et al., 1997 In this paper, we describe an analysis of RDR-mediated defense against viruses in the virological model species Nicotiana benthamiana (Nb). We show that silencing of NbRDR6 causes plants to be hypersusceptible to PVX, potato potyvirus Y (PVY), and the Y satellite of CMV but not TMV, tobacco rattle tobravirus (TRV), turnip crinkle carmovirus (TCV), or CMV alone. The PVX hypersusceptibility was associated with the enhanced viral invasion of the growing point of the infected plant. We also show how NbRDR6 is implicated in systemic RNA silencing; it is not required for production or translocation of the silencing signal but it is required for cells to respond to received signal. By combining our findings about meristem invasion and systemic silencing, we derive a model of virus defense in which NbRDR6 recruits a PVX-derived silencing signal to trigger an immediate silencing response against virus as it enters the growing point and newly emerging leaves. This silencing signal-related mechanism could explain, at least in part, why PVX and possibly other viruses are not able to invade the meristem of infected plants.
Identification of the N. benthamiana Ortholog of AtRDR6 To investigate the role of RDR6 in antiviral defense, we generated an N. benthamiana line in which NbRDR6 was silenced. The silencing construct was based on a 516-bp fragment of N. benthamiana RDR6 cDNA that was PCR amplified using primers corresponding to highly conserved regions in RDR6 of Arabidopsis and other plants (Fig. 1A). The predicted translation product of the amplified DNA sequence is more similar (78.5% identical) to RDR6 than to any of the other Arabidopsis RDR proteins (42.3%, 27.3%, 25.0%, and 27.3% identity with RDR2, RDR3, RDR4, and RDR5, respectively; Fig. 1B), and we conclude that it represents a structural ortholog of Arabidopsis RDR6 henceforth referred to as NbRDR6.
To confirm that NbRDR6 is functionally similar to RDR6, we cloned a 243-bp portion of the original 516-bp NbRDR6 cDNA into a TRV silencing vector. This construct (TRV:RDR6) was inoculated to N. benthamiana in which a green fluorescent protein (GFP) transgene was posttranscriptionally silenced, and, after 4 to 6 weeks, these plants exhibited a breakdown of GFP silencing, similar to the phenotype of RDR6 mutants in Arabidopsis (Dalmay et al., 2000 ). The loss of silencing was manifested as an increase in GFP fluorescence and RNA levels (Fig. 2, A and B). No breakdown of GFP silencing occurred in plants inoculated with the empty TRV vector, showing that this phenotype is caused specifically by the virus-induced silencing of NbRDR6 in TRV:RDR6-infected tissue.
For analysis of NbRDR6 without the complication of TRV infection we generated the N. benthamiana line RDR6i, in which NbRDR6 is constitutively silenced by an RNAi hairpin construct. The GFP16c/RDR6i line additionally expresses GFP under the control of the cauliflower mosaic virus 35S promoter. In quantitative real-time reverse transcription (RT)-PCR analyses the NbRDR6 transcript levels were reduced to 4% of the level in a line carrying an unrelated RNAi construct, targeting the  -glucuronidase (GUS) gene (GFP16c/GUSi), whereas expression of the N. benthamiana orthologs of RDR1 and RDR2 was not affected significantly (Fig. 3). The GFP16c/RDR6i plants phenocopied the Arabidopsis rdr6 mutants; silencing of GFP by a sense GFP construct was impaired, whereas an inverted repeat GFP construct (GFPi) induced local silencing of GFP and formation of GFP-derived siRNA (Supplemental Fig. 1, A and B). GFP silencing by GFPi was equally efficient in GFP16c and GFP16c/RDR6i plants, indicating that expression of the RDR6i construct did not overload and inhibit the silencing machinery (Supplemental Fig. 1C).
Reduced Expression of NbRDR6 Results in Hypersusceptibility to Some Viruses
To investigate the role of NbRDR6 in virus defense (Mourrain et al., 2000
We inoculated RDR6i and nt plants with a PVX:GFP vector and monitored GFP fluorescence to find out whether the RDR6i phenotype was associated with altered virus movement. In inoculated leaves the PVX:GFP infection foci showed no significant difference in size between RDR6i and nt plants (Fig. 4B). However, in the systemically infected leaves, there was a difference in GFP fluorescence that was apparent as soon as 7 dpi. In the RDR6i line, 18/22 plants exhibited GFP fluorescence in noninoculated upper leaves, whereas in nt plants, the GFP fluorescence was weak (12/22 plants) or not detectable (10/22; Fig. 4C). By 12 dpi, the newly emerging leaves of RDR6i plants were all fully green fluorescent, while nt leaves emerged with only a few fluorescent areas (Fig. 4, C and D). The differential accumulation of PVX:GFP in upper leaves of RDR6i and nt plants continued until 17 dpi (Fig. 4C). Corresponding to the differential PVX:GFP accumulation in leaves, the stems of RDR6i were uniformly green fluorescent up to and including the apical growing point, whereas nt stems exhibited no, or very low level, fluorescence in the upper 0.5 to 1 cm (Fig. 4E). A northern-blot analysis confirmed that the symptom and GFP differences correlated with PVX:GFP RNA accumulation. Thus, in inoculated leaves of nt and RDR6i plants, there was between-plant variation in the PVX:GFP RNA accumulation but, overall, there was no difference between the two lines (Fig. 5A). However, in the upper infected leaves, the PVX:GFP RNA was more abundant in RDR6i. This difference was more pronounced at 10 dpi and later when PVX:GFP RNA levels decreased in the upper leaves of the nt plants but remained high in RDR6i (Fig. 5B).
These combined GFP and RNA data indicate that the kinetics of viral RNA accumulation and cell-to-cell spread were the same on inoculated leaves of nt plants and RDR6i (Figs. 4B and 5A). It is therefore unlikely that NbRDR6 influences PVX replication or movement between cells. Instead, it seems that NbRDR6 inhibits PVX accumulation in the growing point and newly emerged leaves. Presumably the RDR6i plants are hypersusceptible to PVX because NbRDR6 normally impairs systemic virus movement or is involved in the mechanism that normally excludes PVX and other viruses from the growing point of the infected plant.
An explanation of the NbRDR6-mediated effect on systemic movement of PVX invokes an RNA silencing signal in PVX-infected plants that would move systemically either with or ahead of the virus (Voinnet et al., 2000 If this hypothesis is correct, RDR6 and the silencing signal would contribute to silencing of viral RNA in cells that are at, but not behind, the systemic infection front. To test this prediction, we monitored PVX-induced silencing in plants either with or without the RDR6i transgene either at or behind the systemic infection front. For analysis of cells behind the infection front, we sampled the third leaf from the top of nt and RDR6i plants at 10 dpi and found no significant difference in PVX:GFP RNA and virus-derived siRNA levels (Fig. 6A). These leaves were further behind the infection front than those used for the analysis in Figure 5 and the absence of a difference in the two lines indicates that RDR6 does not significantly contribute to siRNA production once the infection is established.
However, there was an effect on the silencing signal at the systemic infection front that we observed by comparison of the PVX:GFP-infected lines GFP16c and GFP16c/RDR6i (Fig. 6B; Supplemental Fig. 3). The, young, not yet fully expanded leaves of GFP16c plants (Fig. 6B, leaf 1) exhibited three GFP expression levels indicative of virus- and transgene-derived gene expression. First, there was widespread background GFP fluorescence due to the 35S:GFP transgene (Fig. 6B, arrow 1); second, there were localized spots of intense fluorescence due to strong GFP expression from replicating PVX:GFP (Fig. 6B, arrow 2); and third, in regions around the veins, the GFP fluorescence was lost due to spreading of the silencing signal (Fig. 6B, arrow 3). The GFP16c/RDR6i leaves at the same stage showed more extensive fluorescence due to PVX:GFP than in GFP16c and no evidence for spread of the silencing signal from the veins (Fig. 6B, leaf 1). In older leaves (Fig. 6B, leaf 3), GFP silencing occurred only around infected areas in GFP16c/RDR6i plants but it was more restricted than in the GFP16c plants and did not spread ahead of the virus front alongside the veins. As expected, if a virus-derived silencing signal spreads with or ahead of the virus and prevents virus accumulation, the GFP-silenced tissue in these plants did not subsequently become infected. These results are therefore consistent with a role of RDR6 in either production of the signal or in initiation of silencing in cells that receive the systemic signal. To investigate the silencing signal in more detail, we carried out grafting assays with RDR6i plants. First, we used an N. benthamiana line that carries the GFP16c transgene and a GFP RNAi construct (GFPi). Plants of this line, designated GFP16c/GFPi, are red under UV light at all stages of growth. When GFP16c scions were grafted onto GFP16c/GFPi stock plants, the GFP silencing spread into the scion after 14 d in 24/24 plants (Fig. 7A). In contrast, none out of 24 GFP16c/RDR6i scions on GFP16c/GFPi rootstocks exhibited GFP silencing after 28 d and, of these, 13/13 plants that were kept for a further 60 d remained fully green fluorescent (Fig. 7B). As expected, GFP-derived siRNA was detectable only in silenced GFP16c scions but not in nonsilenced GFP16c/RDR6i scions (Supplemental Fig. 4). In a replicate experiment, the young leaves in six out of 24 GFP16c/RDR6i scions exhibited a pattern of tightly vein-restricted GFP silencing under UV light. However, this limited silencing did not spread further into the mesophyll at later time-points and eventually it faded (Fig. 7C). This suppression of systemic silencing in the scions was a specific effect of the RDR6i transgene because GFP16c scions with a GUS RNAi transgene were fully competent to receive the GFP silencing signal at the same time as GFP16c scions (data not shown). Our conclusion from these grafting experiments is that NbRDR6 influences the ability to respond to a silencing signal that is translocated out of the GFP16c/GFPi rootstocks.
We tested for the production of a systemic silencing signal in RDR6i by transient expression of the GFPi construct in GFP16c and GFP16c/RDR6i rootstocks. Local GFP silencing was triggered in both plant lines (Supplemental Fig. 1, A and B) and systemic spread of GFP silencing into noninoculated leaves occurred in GFP16c plants, although not in GFP16c/RDR6i (Fig. 7, D and E). This failure of systemic silencing signal in GFP16c/RDR6i was not because the silencing signal was absent. In six out of seven GFP16c plants that were grafted as a scion onto these plants, there was systemic silencing after 24 d (Fig. 7E). NbRDR6 is therefore not required for production of the systemic silencing signal. We can also rule out that NbRDR6 is required for transport of the silencing signal from a three-way grafting experiment in which 2-cm long RDR6i stems were grafted between a GFP16c/GFPi rootstock and a GFP16c upper scion. In this experiment, the upper scion, but not the middle one, became silenced in seven out of nine plants, beginning at 31 d after grafting of the GFP16 scions (data not shown). Therefore, from these grafting experiments, we conclude that RDR6 affects systemic silencing because it influences a cell's ability to respond to the silencing signal. There is no evidence that this protein affects production or translocation of the signal.
In this paper, we describe how NbRDR6, like its Arabidopsis homolog, is implicated in a virus defense mechanism (Mourrain et al., 2000 ; Dalmay et al., 2001); plants in which NbRDR6 is silenced exhibit hyper-susceptibility to PVX, PVY and CMV in combination with the Y satellite although not to TRV, TMV, TCV, and CMV alone. With PVX, the antiviral role of NbRDR6 is in the growing point and newly emerging leaves of the infected plant rather than at the level of replication or cell to cell movement of the virus in the inoculated leaf (Fig. 4). We discuss below how this observation and our findings that NbRDR6 is implicated in systemic silencing may be informative about the role of RNA silencing in systemic spread and meristem exclusion of plant viruses.
In principle, NbRDR6 might affect susceptibility to PVX because the effector complex of RNA silencing is not formed in RDR6i. However, this effector complex is fully functional in the absence of NbRDR6, as illustrated by virus-induced and RNAi silencing phenotypes of rdr6 in Arabidopsis (Dalmay et al., 2000
We can also rule out that NbRDR6 is required for systemic signal production (Fig. 7), and a more likely scenario to explain PVX invasion of the meristem in RDR6i plants is based on a proposed role of NbRDR6 in cells that receive the signal. This interpretation is consistent with genetic analysis in Arabidopsis showing that long distance movement of a silencing signal is impaired in rdr6 mutants (Himber et al., 2003
This model (Fig. 8) requires that PVX produces a systemic silencing signal and that this signal spreads through the plant either with or ahead of the virus front. Consistent with this prediction, PVX:GFP produces a signal that travels slightly ahead of the virus front (Fig. 6). The proposal that a signal is produced from PVX:GFP differs from a suggestion that the p25 silencing suppressor protein of PVX prevents long range silencing in grafting assays (Voinnet et al., 2000 ). According to this earlier hypothesis, p25 would prevent all signal production in PVX-infected cells. However, the grafting assay tested for a silencing signal that would travel many centimeters away from the infected cells, whereas our present hypothesis requires only that the signal is at or slightly ahead of the virus infection front. The signal we observed in PVX:GFP-infected GFP16c plants (Fig. 6B) could have been generated inside the vasculature in such amounts that p25 is not sufficient to stop it from moving short distances and spreading into the mesophyll.
A silencing signal-based mechanism of virus defense could operate anywhere at the infection front. However, it might be expected that this process would be particularly important in the growing point. This part of the plant is a strong photosynthetic sink and, consequently, a preferred transport destination of both viruses and silencing signals. In effect, this silencing signal hypothesis proposes that the well-established phenomenon of meristem exclusion (Matthews, 1991
Further supporting evidence that silencing is involved in meristem exclusion is from the previous findings that RNA viruses acquire the ability to invade meristems if they are inoculated to plants expressing viral suppressors of silencing (Foster et al., 2002
It is not clear at present why RDR6i plants are not hypersusceptible to all tested viruses. Only PVX, PVY, and CMV in combination with its Y satellite exhibited enhanced virulence on RDR6i plants (Fig. 4; Supplemental Fig. 2). Perhaps the signal from the cells infected with TMV, TRV, and other viruses is produced at too low levels or too late to have an effect on virus spread. Alternatively, the RNA of the other viruses may have sequence elements or structures that prevent its being used as a template by NbRDR6. There are five other RDR proteins in Arabidopsis and it may be that one or more of them use other viral RNAs. Consistent with this idea, the ortholog of RDR1 has been shown to contribute to defense against TMV and PVX in N. tabacum (Xie et al., 2001 For a full understanding of RNA silencing in antiviral defense, it will be necessary to generate plants that are mutant or silenced for the remaining RDR homologs and also for other genes including AGO and DCL homologs that are required for RNA silencing. Our analysis described here shows how the effects of these proteins on virus susceptibility will need to be assessed in the whole plant rather than simply at the level of the cell.
Transgenic Plants and DNA Constructs
Nicotiana benthamiana line GFP16c has been described as line 16c previously (Ratcliff et al., 2001
The same fragment was also cloned blunt-ended into the SmaI site of a TRV-based gene silencing vector (Jones et al., 1999
To obtain construct GF:invTerm, the octopine synthase terminator from construct GFPi was amplified using primers 5'-ATCCGTCACTACGTGTAGTCCCTAGAGTCCTGTC-3' and 5'-TGCATCCACGTAGTGCAGTCACGACGTTGTAAAAC-3', cloned into the DraIII site of construct 35S:GF (Lu et al., 2003 For transient expression in N. benthamiana, pGreen- and pBin-based constructs were mobilized into Agrobacterium strain GV3101 (containing the pSoup helper plasmid) or C58C1 (pCH32 helper plasmid), respectively.
All plants were grown in a glasshouse with 16-h supplemental lighting (HQI halide lights) at a constant temperature of 22/20°C (day/night).
Inoculation of N. benthamiana with Agrobacterium carrying constructs for transient expression of transgenes or viruses was done as described previously (English et al., 1997 For the assessment of NbRDR6 function in N. benthamiana, 2- to 3-week-old plants of line 16c were systemically silenced for GFP by agro-inoculating GFPi. Six weeks postinoculation, when the plants were completely silenced and appeared red under UV light, lower leaves were agro-inoculated with TRV:RDR6 and upper, noninoculated leaves were monitored for breakdown of GFP silencing under UV light, which usually appeared 4 to 6 weeks postinoculation with the VIGS vector in all five to 10 inoculated plants.
For grafting of N. benthamiana, 3- to 4-week-old plants were used. Scion stems were cut to a wedge shape that was then inserted into a vertical slit cut into the stem of the rootstock about 2 cm above soil level. The grafting junction was wrapped with Parafilm and plants were kept humid under a plastic cover for 1 week or until the grafts had taken.
GFP expression in plants was photographed under UV light using a Nikon D1X digital camera with a Kodak Wratten filter number 8 and a B-100AP longwave-UV lamp (Ultra-Violet Products, Upland, CA) or using a Leica MZ-FLIII dissecting microscope with GFP-filter and a Leica DC200 digital camera (Leica, Solms, Germany).
Total nucleic acid was extracted from plant tissue by either using TRI Reagent (Sigma, St. Louis) or, for viral RNA analyses, following the procedure of White and Kaper (White and Kaper, 1989 For the detection of siRNAs, equal amounts of total RNA (approximately 10 µg) were separated on 8% polyacrylamide gels in 1x Tris-borate/EDTA with 50% (w/v) urea and transferred onto Zeta-Probe GT membranes (Bio-Rad, Hercules, CA) by overnight capillary transfer in 20x SSC. In vitro transcribed 32P-labeled RNA probes of cloned fragments of GFP, GUS, RDR6, or the PVX-coat protein sequence were used to detect corresponding siRNA over night at 42°C using PerfectHyb hybridization buffer (Sigma) following a 2-h prehybridization in the same buffer.
Viral RNA was detected by separating 5 µg total nucleic acid on formaldehyde agarose gels and transferring to Zeta-Probe GT membranes (Bio-Rad) by overnight capillary transfer according to standard laboratory protocols (Sambrook and Russel, 2001
Transcript levels of RDR genes were analyzed by quantitative real-time RT-PCR using the Chromo4 detector in combination with a PTC-200 Thermal Cycler (MJ Research/Bio-Rad). Total RNA was extracted from four pools of five plants at six-leaf-stage for each plant line using TRI Reagent (Sigma) according to manufacturer's instructions. Synthesis of cDNA from 5 µg of total RNA was performed with random hexanucleotides (Roche, Mannheim, Germany) and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) following manufacturer's instructions. Reactions without reverse transcriptase or without template were included as controls. For the quantitative real time PCR, cDNA corresponding to 500 ng of total RNA was used in 50-µL reactions using DyNAmo HS SYBR Green qPCR kit (Finnzymes, Espoo, Finland) according to manufacturer's instructions. Primers were designed to amplify similar sized regions of GAPDH, serving as an internal standard, and the N. benthamiana orthologs of RDR6, RDR1 (GenBank accession no. AY574374; Yang et al., 2004 Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number DQ093875.
The authors thank Louise Chappell for CMV Y satellite inoculations and images, Attila Molnar and Natalya Elina for critical reading of the manuscript, and Sharyn Carter for excellent technical assistance. Received March 30, 2005; returned for revision April 20, 2005; accepted April 20, 2005.
1 This work was supported by the European Commission Research Directorate (contract no. QLG2–CT–2002–01673). The Sainsbury Laboratory is supported by the Gatsby Charitable Foundation. 
2 Present address: Department of Biology, University of York, York YO10 5DD, UK.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.063537. * Corresponding author; e-mail david.baulcombe{at}sainsbury-laboratory.ac.uk; fax (44)(0)1603–450011.
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